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First published online November 10, 2004
doi: 10.1242/10.1242/jcs.01538

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The case for nuclear translation

¹ MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DS, UK
² Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, PO Box 88, Manchester, M60 1QD, UK
³ The Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK

View larger version (32K): [in a new window]   Fig. 1. Nonsense-mediated decay. (A) The phenomenon. The gene (with promoter, parts of the first and penultimate exons, and last exon), primary transcript, spliced mRNA (with an exon junction complex – EJC – deposited 5' of the exon-exon junction) and protein are shown. UPF proteins associate with the EJC (not shown). (i) Transcription of a gene with a termination codon in the correct place leads to a spliced and stable mRNA, and a full-length protein. (ii) Moving the termination codon 5' to what will become an exonexon junction leads to an unstable mRNA and a truncated protein. (B) Models for NMD. In each case, an mRNA encoding a PTC is made at the transcription site by a polymerase (pol). The mRNA with its EJC then passes through the nucleoplasm to dock at the membrane, before exiting through the pore to the cytoplasm. The mRNA is destroyed once the NMD machinery detects the PTC. (i) A cytoplasmic ribosome detects the PTC, and transmits a signal to the nucleus that leads to degradation of homologous transcripts containing the PTC. (ii) NMD occurs as the transcript bearing the PTC emerges into the cytoplasm. (iii) Some unknown nuclear mechanism (black box) recognizes the PTC and destroys the mRNA. (iv) A ribosome detects the PTC in the nascent transcript at the transcription site.

View larger version (73K): [in a new window]   Fig. 2. Poly(A)+ RNA in the nucleus and cytoplasm. Little poly(A)+ RNA is found at the nuclear periphery in a mouse fibroblast (NIH 3T3). Poly(A)+ RNA was detected by in situ hybridization using biotinylated poly(dT)54 and streptavidin conjugated with Alexa594 (pseudo-coloured green); DNA was counterstained with DAPI (blue). Image kindly provided by Meg Byron, John McNeil and Jeanne Lawrence. Bar, 10 µm.

View larger version (178K): [in a new window]   Fig. 3. The nuclei (but not cytoplasm) of permeabilized HeLa cells exclude a 40 kDa dextran conjugated with fluorescein isothiocyanate (FITC). Hela cells were permeabilized in saponin in a `physiological' buffer, washed in the same buffer, incubated (15 minutes, 27.5°C) in the precursors required for translation (as for Fig. 2 of Iborra et al., 2001), a 40 kDa dextran-FITC added, and cells imaged on a confocal microscope. FITC fluorescence is illustrated. Bar, 10 µm.

View larger version (27K): [in a new window]   Fig. 4. A model for transcript production (Iborra et al., 2004). (A) The CTD has the potential to associate with sites involved in capping, transcript degradation, translational proofreading (involving the translational and NMD machineries), proteolysis, splicing and polyadenylation. It remains unclear whether all bind to the CTD simultaneously, or whether they attach and detach as needed. [Wetterberg et al. (Wetterberg et al., 2001) have analysed the association of the splicing machinery during the synthesis of an exceptionally long mRNA.] (B) Transcription began as the template bound to the polymerizing complex and was reeled in as the transcript was extruded; the CTD is now hyper-phosphorylated (CTDP), and a cap has been added. (C) The transcript continues to be extruded through a splicing site as the ribosome/NMD machinery begins proofreading the now-spliced message (and so does not read introns, which might contain many termination codons). (D) Once introns are removed (lariat), the transcript is cleaved and polyadenylated, and is ready to leave for the cytoplasm. If errors are detected, the faulty transcript and peptide produced during proofreading are degraded by nucleases and proteasomes.